The associations of age-related macular degeneration (AMD) with cataracts, prior cataract surgery, cumulative exposure to sunlight and pigmentation all support the hypothesis that chronic photochemical injury drives macular changes with age and AMD progression. Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) and colocalizes with acute photosensitization of reactive oxygen intermediates (ROI) in the primate retina. We model the normal accumulation of potentially damaging photoproducts with age in the RPE and Bruch?s Membrane (BM) complex as well as changes induced by additional spectral filtering of light reaching the macula. Lipofuscin granules contain at least 10 different fluorescent photochemical products includng A2E (N-retinylidene-N-retinylethanolamine), its epoxides and other as yet chemically unidentified A2E-related fluorophores. The precursors (A2PE and A2PEH2) of these fluorophores originate from reactions of all-trans-retinal within the receptor outer segments (ROS) during periods associated with significant rhodopsin bleaching (i.e., normal daylight). Although RPE lysosomal processing enzymatically digests over 99% of the shed ROS contents, A2E and related fluorophores are not digested, but are concentrated into lipofuscin granules. By age 60 years, the average concentration of A2E within RPE cells reaches ~400 microM in normal eyes. However, A2E is toxic to cellular membranes at much lower concentrations. We hypothesize that segregation of A2E into lipofuscin granules and prevention of its redistribution into critical membranes is required for RPE health. [unreadable] [unreadable] We developed a biophysical model using normal values of pupil size, lens transmission, and rod dark adaptation time constant trh to determine average retinal spectral irradiance, steady-state concentration of all-trans-retinal, all-trans-retinal photosensitization of oxidative damage, all-trans-retinal reactions to form A2E-related species in the ROS, and A2E photo-oxidation within RPE lipofuscin granules as a function of age and ambient light intensity. Our model predicts a decline of about one third in the action spectra-weighted short-wavelength macular irradiance with each decade and a nearly constant production rate of A2E-related fluorophores in the RPE during the first 60 years (falling significantly thereafter). A similar age dependence of total lipofuscin granule volume and total fluorescence per RPE cell was reported recently in human cadaver eyes. Since the rates of lipofuscin increase with age are slower than the rate of decrease in short-wavelength macular irradiance in the phakic eye with age, ROI photosensitization in the RPE should also fall with increasing age. Photo-oxidative stress in the outer retina might arise from the smaller amounts of A2E-related fluorophores observed in critical membranes of the RPE/BM complex. However, if the RPE/BM complex were the site of photo-oxidative injury driving AMD progression, the magnitude and rate of this oxidative injury would be expected to increase dramatically (not observed) following cataract removal and intraocular lens (IOL) implantation. [unreadable] [unreadable] Consequently, we propose a novel hypothesis that singlet oxygen generation by RPE lipofuscin allows the chemical alteration of accumulating A2E, thereby limiting the steady-state levels of A2E ([A2E]ss) in the RPE, the redistribution of A2E into retinal membranes, and associated A2E chemical toxicity. Singlet oxygen generated photochemically within the lipofuscin granule reacts with its A2E to form A2E epoxides which then react to form increasingly complex cross-linked molecules. As short-wavelength macular irradiance falls with age, the rate of A2E photo-oxidation falls approximately up to 20-fold, causing [A2E]ss in the normal phakic eye to increase even as rod bleaching and A2E production decrease. Our theoretical model of macular aging reproduces the normal age dependence of lipofuscin and A2E and provides a primary cytotoxic mechanism in which, once A2E exceeds a cytotoxic threshold concentration in the RPE cell, A2E redistribution into critical membranes causes damage and loss of RPE function with or without additional photo-activation. In our model, it is primarily the yellowing of the lens with age that distorts the original spectral balance between rate of production and rate of photo-oxidation found in youth, and allows the [A2E]ss to rise with age.[unreadable] [unreadable] We are evaluating noninvasive retinal imaging methods that might permit clinical validation of our predictions of photochemical changes following cataract surgery and our predictions of the benefits of specific spectral photo-protective filters. We have designed and had manufactured "bicolored" sunglasses with two different specific spectrally selective lenses that provide equivalent spectral photoactivation of lipofuscin photosensitization of singlet oxygen but very different degrees of rod activation in bright ambient light. Our model predicts that in young eyes or elderly pseudophakes the levels of A2PEH2 production in the rods and the [A2E]ss levels in the RPE will be remarkably divergent in individuals wearing these sunglasses over a period of a few months of significant outdoor daylight activity. In order to distinguish the steady-state levels of different RPE photoproducts in the retina noninvasively, we are developing spectral imaging capabilities by modifying confocal scanning laser ophthalmoscopes and fundus cameras. We are currently evaluating the potential of these modified clinical instruments for noninvasive monitoring of the molecular effects of such filters in patients. Furthermore, we expect that improved spectral separation of the different species in the "A2E pathway" along with high resolution noninvasive imaging of the human retina, should enable us to quantify early microscopic abnormalities that we believe are the earliest stages in age-related maculopathy leading to AMD as well as genetically -linked diseases such as Stargardt's macular dystrophy. Such quantitative early intermediate endpoints may prove critical in developing new more efficient clinical studies of AMD prevention. In collaboration with the NEI and the Eye Institute of the Russian Academy of Medicine, we are designing clinical studies of the effects of such filters on progression of both early and moderate AMD following cataract surgery and IOL implantation and macular changes in subjects with genetic predisposition to generation of increased A2E and lipofuscin at younger ages.[unreadable] [unreadable] In parallel laboratory experimentation, we are studying RPE cell cultures, human autopsy eyes, and animal models to better characterize the pathways and rates of A2E photo-oxidation and chemical modification. In collaboration with our sister NICHD lab (Steve Coon), we are planning to test our hypothesis of spectral imbalance by raising ABCA4 (-)/(-) and (-)/(+) knockout mice (that produce large amounts of A2E) in cyclic ambient light spectrally filtered eitehr with the rod-sparing vermilion filter or the amber filter which does not spare rods but provides the equivalent amount of protection from blue-light photo-oxidation reactions within the retina. This model system should validate our noninvasive spectral imaging separation of components by comparing with HPLC-MS of lipofuscin extracts from the whole retina at sacrifice and provide a stronger experimental rationale for our proposed clinical studies by validate the basic hypothesis that spectral imbalances of retinal irradiance can induce high A2E steady state levels in teh RPE.